The term "Neisseria gonorrhoeae" as used herein, refers to the bacteria classified as such in Bergey's Manual of Systematic Bacteriology (N. R. Krieg [ed.], 1984, pp. 288-296, Williams and Wilkins). Detection of Neisseria gonorrhoeae is important in various medical and public health contexts. Neisseria gonorrhoeae is one of the leading causes of epidemic sexually transmitted disease, with approximately one million cases reported in the United States in 1983. Infection with this pathogen can result in a wide variety of clinical manifestations, most commonly urethritis, cervicitis and proctitis. However, infection frequently results in diseases requiring hospitalization, such as endometritis, salpingitis and pelvic inflammatory disease.
Therefore, it is an aspect of the present invention to provide nucleic acid probes for use in a hybridization assay system capable of rapidly detecting Neisseria gonorrhoeae and which is generally applicable to all types of clinical samples.
The scope and severity of disease caused by this organism have resulted in the development of a variety of methods for detection from clinical samples, however, the only methods currently recommended by the Center for Disease Control, the Public Health Association or the American Society for Microbiology for detection of this organism from male or female specimens rely primarily on culture.
Highest culture recovery of viable organisms requires immediate plating of a freshly collected specimen on an appropriate selective culture medium (e.g. Thayer-Martin medium) and growth in a reduced oxygen atmosphere (3-10% CO.sub.2) at 35.degree. C. If immediate plating is not possible, then a non-nutritive transport system can be used, as long as the sample is held for less than six hours. If the sample cannot be plated within six hours, then a nutritive transport system can be employed, containing growth media and a source of CO.sub.2. Following plating on selective media, cultures are incubated at 34.degree.-36.degree. C. in a 3%-10% CO.sub.2 environment for 28-48 hours. Colonies suspected of being Neisseria then are Gram-stained and tested for oxidase activity. Oxidase positive, Gram-negative diplococci are indicative of Neisseria spp. and must then be confirmed as Neisseria gonorrhoeae, since the culture sites may contain a variety of non-pathogenic Neisseria. Confirmation can be carried out in a variety of ways, most commonly by carbohydrate utilization, latex agglutination or immunofluorescence tests. However, any of these confirmatory tests are labor intensive and time consuming, often requiring additional incubation and/or growth periods.
It is another aspect of the present invention to avoid the disadvantages associated with traditional culturing techniques.
An enzyme immunoassay for identification of Neisseria gonorrhoeae directly from clinical specimens (Gonozyme.TM., Abbot Laboratories, Chicago, IL) has been available for several years, however, a variety of clinical studies have indicated that this test suffers from a lack of sensitivity and specificity. The assay also requires 3-4 hours to perform, and requires the purchase of specific signal detection equipment.
It is yet another aspect of the present invention to avoid the disadvantages associated with enzyme immunoassays and to employ nucleic acid probes to detect Neisseria gonorrhoeae.
It is still another aspect to provide nucleic acid probes and hybridization techniques are described which permit the specific detection of Neisseria gonorrhoeae in clinical specimens.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially, see below--Hybridization) to target nucleic acid sequences.
Hybridization traditionally is understood as the process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds.
Totten et al. (The Journal of Infectious Diseases, 1983, 148:462-471) describe detection of Neisseria gonorrhoeae in clinical specimens by utilizing DNA probes directed against the so-called cryptic plasmid which commonly is associated with this bacterium. However, it is well known that the presence of this plasmid in Neisseria gonorrhoeae isolates is highly regional (occurring in from 78% to 98% of isolates in different parts of the United States), predicting a high degree of variability in any assay based on the detection of its presence in test samples.
It is yet another aspect of the present invention to remove this source of variability by providing probes which hybridize to nucleic acid sequences that are common to all strains of Neisseria gonorrhoeae, but which do not hybridize to any commensal non-Neisseria gonorrhoeae or other flora that may be present in test samples.
Lo and Yang et al. (European Patent Application 87101215.9) describe the isolation of nucleic acid probes directed against chromosomal genes of Neisseria gonorrhoeae. These probes are purported to specifically recognize six strains of Neisseria gonorrhoeae, with no cross-hybridization to six strains of Neisseria meningitidis. Welcher et al. (Nucleic Acids Research, 1986, 14:10027-10044), also describe the isolation of chromosomally-targeted DNA probes. These probes are purported to specifically recognize the strain from which they were selected as well as six clinical isolates of Neisseria gonorrhoeae, with no cross-hybridization to 7 other Neisseria spp., E. coli or the commensal bacterium Branhamella catarrhalis. However, testing of the aforementioned probes against a wide variety of organisms was not reported. Also, because the chromosomal target sequences are represented in low copy number in each cell, the described assays are not very sensitive and, as a consequence, their utility is significantly restricted in clinical applications.
It is yet another aspect of the present invention to provide nucleic acid probes which combine high specificity for Neisseria gonorrhoeae with high sensitivity by the utilization of probes which hybridize to ribosomal RNA molecules which are present in high abundance in the target bacteria.
Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Ribosomes contain three distinct RNA molecules which, at least in E. coli, are referred to as 5S, 16S and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary somewhat in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacterium, and this convention will be continued herein.
While Kohne et al. (Biophysical Journal 8:1104-1118, 1968) discuss one method for preparing probes to rRNA sequences, they do not provide the teaching necessary to make Neisseria gonorrhoeae-specific probes.
Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantifying such homology levels. Similarly, Sogin, Sogin and Woese (Journal of Molecular Evolution 1:173-184, 1972) discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships. Fox, Pechman and Woese (International Journal of Systematic Bacteriology 27:44-57, 1977) discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systematics. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to Neisseria gonorrhoeae and, in particular, do not provide Neisseria gonorrhoeae-specific probes useful in assays for detecting Neisseria gonorrhoeae in clinical samples.
Hogan et al. (European patent publication WO 88/03957) describe a number of probes which are claimed to be specific for Neisseria gonorrhoeae rRNA. However, Hogan et al. only demonstrate that their probes hybridize to the Neisseria gonorrhoeae strain against which they are designed-a foregone, and hardly novel, conclusion. No data is given to suggest that any of the probes will hybridize to additional N. gonorrhoeae strains. Similarly, the specificity of the probes is defined by lack of hybridization to only single strains of a few (8) related Neisseria species. No data is provided to indicate which, if any, of the probes have the claimed specifity, i.e. that they are inclusive for all or even most N. gonorrhoeae.
It is another aspect of the present invention to provide nucleic acid probes that are well characterized with respect to their hybridization behavior toward Neisseria and non-Neisseria bacteria.
Ribosomal RNAs are highly structured molecules. This structure depends largely on the same types of interactions which govern probe-target interactions. Therefore, not all potentially useful hybridization target sequences are equally accessible to probes under every conceivable assay condition.
It is yet another aspect of the present invention to provide probes and probe sets which can hybridize to rRNA target regions which can be rendered accessible to probes under normal assay conditions.
The stringency of a particular set of hybridization conditions is defined by the length and base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids.
Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.
As a general matter, dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree. C.-65.degree. C. in a salt solution of approximately 0.9 molar.